Droplet Digital Polymerase Chain Reaction (PCR) Outperforms Real-Time PCR in the Detection of Environmental DNA from an Invasive Fish Species

Abstract

Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors.

A Circulating microRNA Signature Predicts Age-Based Development of Lymphoma

“…ddPCR has superior precision over other detection technologies to detect differential expression in miRNAs [39]. Additionally, ddPCR is more resilient to sample quality differences; has a high tolerance to PCR amplification inefficiency; and requires small sample size, which allows for high level of precision being able to capture all known sources of variation [39, 40]. …”

Controls on eDNA movement in streams: Transport, Retention, and Resuspension

“…At higher concentrations (>200 copies/μL), recent research has shown that qPCR and ddPCR are equivalent at detecting eDNA 37 however, ddPCR can detect eDNA significantly better at very low concentrations (<3 copies/mL) 12, 25, 38 . Using the same primers and Taqman probe from qPCR, target DNA was randomly allocated on to discrete droplets using microfluidics and then each of the samples, containing 10,000 to 20,000 nanodroplets via microfluidics that are then thermally-cycled (see Nathan et al .…”

Isopropanol precipitation method for collecting fish environmental DNA

“…In the pond experiment, we failed to find any significant differences in the detection rate of carp eDNA by the two precipitation methods, when the reaction volume was the same, perhaps because the detection rate was only determined by the presence or absence of a few copies of target eDNA (Doi et al ). However, in samples with low eDNA concentrations, the isopropanol precipitation method would exhibit a higher detection rate, owing to a higher amount of recovered eDNA.…”

“…The environmental DNA (eDNA) method can be used to estimate the distribution of aquatic organisms, using DNA extracted from the water of aquatic habitats (Ficetola et al ; Rees et al ; Goldberg et al ; Thomsen and Willerslev ). Since the eDNA method is an easy‐to‐use approach, which only requires collecting water from the field, it has been applied to diverse species in various ecosystems, including ponds and lakes (e.g., Ficetola et al ; Thomsen et al ; Jerde et al ; Takahara et al ; Doi et al ; Uchii et al ), rivers (Minamoto et al ; Pilliod et al ; Fukumoto et al ; Wilcox et al ; Yamanaka and Minamoto ), and marine habitats (Mahon et al ; Miya et al ; Yamamoto et al ).…”